ABSTRACT Precise regulation of blood supply across different tissues is a crucial role of the arterial vasculature. As metabolic rates of tissues change, blood supply shifts accordingly to match oxygen supply to demand in specific locations. In extreme conditions such as heavy exercise, the rate of blood supply to skeletal muscles can increase by over an order of magnitude, a phenomenon possible in large part due to local arterial dilation. Recent research has identified an important role for nitric oxide (NO) signaling in the vascular wall to promote this vasodilatory response. The source of this NO is still hotly debated, as none of the known enzymatic or redox pathways of NO production are believe to create sufficient NO to cause the observed effects. Our laboratory?s discovery of hemoglobin alpha (Hba) expression in the endothelial cells of resistance arteries offers a solution to this contentious question. We hypothesize that when deoxygenated, this spatially optimized pool of Hba reduces nitrite to NO, which diffuses to the adjacent smooth muscle layer to cause vasodilation. Preliminary data indicates that Hba expressed in vascular endothelial cells is a regulator of exercise performance. Aim 1 of this proposal will test the role of endothelial Hba deletion on exercise capacity in vivo. Aim 2 of this proposal will use pressure myography to determine whether endothelial expression of Hba regulates the vasodilation of ex vivo resistance arteries in response to hypoxia. Aim 3 will measure the production of NO in ex vivo resistance arteries in response to hypoxia. Our application presents novel insights into vascular biology, exercise physiology, and has important relevance to multiple fields of cardiovascular medicine. Our research will inform translational applications including stroke, myocardial infarction, pulmonary hypertension, organ transplantation, ischemia/reperfusion injury, and sickle-cell anemia. Our findings will elucidate an emerging mechanism of NO production through which to approach novel NO-based treatments in cardiovascular medicine.